Orange bagasse as biomass for 2G-ethanol production = Bagaço de laranja como biomassa para produção de etanol-2G

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(1)ALMAS TAJ AWAN. ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. BAGAÇO DE LARANJA COMO BIOMASSA PARA PRODUÇÃO DE ETANOL-2G. CAMPINAS 2013. i.

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(3) UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE QUÍMICA. ALMAS TAJ AWAN ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Orientadora/ Supervisora: PROFA. DRA. LJUBICA TASIC. BAGAÇO DE LARANJA COMO BIOMASSA PARA PRODUÇÃO DE ETANOL-2G Tese de Doutorado apresentada ao Instituto de Química da Química da Universidade Estadual de Campinas para obtenção do título de Doutora em Ciências. Doctoral thesis presented to the Institute of Chemistry, University of Campinas to obtain Ph.D. grade in Sciences.. ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA ALMAS TAJ AWAN E ORIENTADA PELA PROFA. DRA. LJUBICA TASIC. ___________________ Assinatura da Orientadora. CAMPINAS 2013. iii.

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(6) DEDICATION My Parents, Malik Taj M. Khan and Zainab K. Awan, for their love, support and encouragement in every phase of my life.. vii.

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(8) Don’t Quit! When things go wrong as they sometimes will; When the road you’re trudging seems all uphill; When the funds are low, and the debts are high; And you want to smile, but you have to sigh; When care is pressing you down a bit Rest if you must, but don’t you quit. Success is failure turned inside out; The silver tint of the clouds of doubt; And you can never tell how close you are; It may be near when it seems afar. So, stick to the fight when you’re hardest hit It’s when things seem worst that you must not quit. Anonymous. ix.

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(10) ACKNOWLEDGEMENTS First of all, I would like to thank God, the Almighty, for making everything possible by giving me strength, courage, wisdom, health, good resources, loving family, precious friends, caring supervisor and everything that made me accomplish this task. I would like to express my deepest gratitude to my Ph.D. Supervisor, Prof. Dr. Ljubica Tasic, for her excellent guidance, care, patience, and providing me with an excellent atmosphere for doing research. Buba, I learned a lot from You and I am simply proud of You, being my Ph.D advisor. Also, I would like to thank Dr. Nelson Duran, who always tried to maintain a healthy atmosphere of LQB lab, sharing his life experiences during travelling and also wonderfull ideas in research line. Special thanks for Prof. Carlos Roque to provide me an opportunity to come from Pakistan and start work in his lab on Organic Synthesis. Many many thanks for my thesis evaluation committee (Prof. Dr. Mahendra Kumar Rai, Prof. Dr. Peter Rudolf Seidl, Prof. Dr. Nelson Eduardo Durán Caballero and Prof. Dr. José Augusto Rosário Rodrigues) for considering, encouraging, commenting and figuring out important areas for improving my written work and thesis. My humble regards to The World Academy of Science (TWAS) and Brazilian National Council for Scientific and Technological Development (CNPq) for supporting my PhD studies in Brazil. Also, I would like to thank Post-graduate coordinator at Institute of Chemistry, University of Campinas to approve me as a PhD Candidate. Some people come and leave footprints on our lives, fortunately during my four years of stay in Brazil, there were a number of people. They are so many, that it is difficult for me to write my words for all of them. But I will try to appreciate in particular those who were related to my academic area. First of all, Juliana Fattori, Sebastian Robles and Gabriel Curti for patiently correcting my Portuguese writing for annual reports. Pablo, Marla, Gago, Patricia, Jason, Ilton, Nilton, Airton, Jailton and Angelica from LASSO lab for their help and friendship during my lab hours and PhD qualification exam. Thanks to Junko Tsukamoto for always being there for. xi.

(11) guidance in my research just like a co-supervisor. I would also like to thank Ana Paula Espindola for taking interest in my work either its written or practical and putting good suggestions. Special thanks for Fabian for staying late hours in lab and helping out in my thesis. Also, Jennifer for being my cute friend and always offering me to go for volleyball, basketball, kungfu and running (and I was always trying to escape). Chico was my only enemy in Brazil who with anti-pakistani and anti-usama jokes always made me laugh. Thanks to two little angels and my youngest Brazilian friends (kids of Buba) Alex and Aninha for taking me back to my childhood while playing with them. Many many thanks for all LQB lab member, Dani Pott, Ana Paula, Junko, Fabian, Daniela, Lilian, Camila, William, Fabio, Isabella, Alessandra, Dani, Jessica, João, Elias, Alzira, Stephany, Lucas, Priscyla, Ana Maria, Lari (I hope I didn’t forget anyone). Thanks for all the technical assistance that was provided by Ricardo in HPLC analyses of my samples, Renata for Infrared analysis, Daniel for SEM analyses, Rita for GC-MS, Anderson and Fabian during my NMR analyses. My special thanks and love for Prof. Adriana Vitorino Rossi for always taking care of me and Surraya as our Brazilian Mom. My gratitude to Prof. Marcia and Maria Helena for helping me during my painting classes. I will also not forget to mention my friends from IQ Bruna, Tiago, Felipe, Francine, Diana, Carol, Leticia, Daniella, Adriana, Eva, Lilian, Marta, Acacia, Edison, Marcelo, Celio and Paulo Filgueiras. Also, my Pakistani friends Surrayya and Fozia, Indian friends Anamika and Rashmi, Irani friend Pooyii, Colombian friends Monica, Sofia, João, Sebastian and my great sister Iram who were always there to celebrate special occasions with me, not letting me feel lonely without family. I would like to thank my family including my younger sisters (Iram, Kiran, Masooma and Urooj), my younger brother (Zeeshan), my brother-inlaw (Sarosh) and my best friend Nasima. I am very grateful to Dr Shahzad Mughal for his support and encouragement to move ahead for doctoral and post-doctoral research. Finally, I would like to thank my parents Malik Taj and Zainab Awan for always being there to cheer me up and standing by my side through the good times and the bad, I love you both and pray for your healthy and long life, ameen.. xii.

(12) Curriculum Vitae ALMAS TAJ AWAN ACADEMIC DEGREES. 2009-2013: Doctor of Chemistry Chemistry Institute, University of Campinas, Sao Paulo, Brazil Project: Orange bagasse as biomass for 2G-ethanol production Supervisor: Prof. Dr. Ljubica Tasic Funding agency: CNPq Fellowship program: TWAS-CNPq post-grad fellowship 2005-2007: Master of Chemistry Institute of Chemical Sciences University of Peshawar, Pakistan Project Supervisor: Prof. Dr. Fazal Mabood Project: Selection of appropriate analytical techniques in compositional analysis PUBLICATIONS . “Supramolecular catalysis” (a review) Authors: Almas Taj Awan, Suryyia Manzoor, Prof. Vanderlei Inácio de Paula Brazilian Journal: Revista Engenho, vol. 6 – september 2012 Available online at http://www.anchieta.br/unianchieta/revistas/engenho/pdf/revista_engenho06.pdf. ORAL PRESENTATIONS IN INTERNATIONAL CONFERENCES Title: Second generation ethanol from orange bagasse via co-culture fermentations Global Biofuels & Bioproducts Summit 19th -21st Nov, 2012 Hilton San Antonio, Texas, USA. Title: Orange Processing Waste as Biomass in Ethanol Green-Production The International Conference, Nanotechnology,Biotechnology and Spectroscopy: Tools of success in the coming Era. ICNBS 1st -3rd March, 2012, Cataract Pyramid Resort Cairo, Egypt.. xiii.

(13) POSTERS PRESENTED IN SCIENTIFIC EVENTS . Title: Second generation ethanol from orange bagasse via co-fermentations Global Biofuels & Bioproducts Summit 19th -21st Nov, 2012 Hilton San Antonio, Texas, USA. . Title: Acidic Hydrolysis of Orange peel for Ethanol Production PIBIC Congress, 24th Oct, 2012 State University of Campinas, São Paulo, Brazil. AWARDS AND RECOGNITION . TWAS 1st prize winner award in SUSTAINABLE ENERGY PHOTO CONTEST Contest on Project’s Photo Theme ‘Sustainable energy for All’ International Week of Science, November 2012 (Italy).. . Inova 2012 UNICAMP Award for Scientific innovation PIBIC Congress; 24th Oct, 2012; Sao Paulo, Brazil.. . Best Oral presentation Award In an International Conference, Egypt The International Conference, Nanotechnology,Biotechnology &Spectroscopy: Tools of success in the coming Era. ICNBS 1st -3rd March, 2012, Cataract Pyramid Resort, Cairo, Egypt.. . Winner of All Pakistan Quaid Conference, 2007 12-13th August, 2007; Baqai Medical University, Karachi, Pakistan.. . Best Debater University of Peshawar, 2006-07 Recognition by Vice Chancellor, University of Peshawar, Pakistan.. EXTRA CURRICULAR  2012-PED-C: Organic Chemistry Laboratory and graduate teaching Assistance; Course Supervisor: Prof. Dr. Fernando Coelho, University of Campinas, Brazil.  2007-2008-Pakistan Television Corporation: Anchor TV shows  2005-2008-Pakistan Radio Broadcasting: Anchor and Producer Radio programs.. xiv.

(14) Abstract: ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION Second generation biofuels from renewable resources have come forth as a result of energy security coupled with diminishing fossil fuel resources. Lignocellulosic biomass is a renewable resource, which can be converted in to liquid transportation fuels. Utilization of agro-industrial waste for the generation of biofuels makes it a cleaner production (Green Chemistry). Brazil is the world’s largest producer of oranges. The current project deals with Citrus Processing Waste from Oranges (CPWO), and obtaining valuable products such as bioethanol, hesperidin, and essential oil. The process of hydrolyzing CPWO was improved and the classical way of biomass saccharification, i.e. acid hydrolysis, was compared with the enzyme hydrolysis. In enzyme hydrolysis, apart from applying commercial enzymes, saccharification was also investigated with protein extracts of Xanthomonas axonopodis pv. citri strain 306 (Xac 306), a potent pathogen that causes Citrus canker disease. Later, the obtained reducing sugars were converted into bioethanol by submerged mono- and co-culture fermentations that involved three yeast strains: Saccharomyces cerevisiae, Candida parapsilosis IFM 48375 and NRRL Y-12969, the last two being isolated from bagasse. Results demonstrated successful hydrolyses by Xac enzymes that released high levels of fermentable sugars. Also during co-culture fermentation processes, it was noticed that ethanol yield was improved from 50% to 62% w/w (calculated on the basis of total dry matter contents) and sugars were consumed faster. Thus by employing co-culture fermentation strategy, apart from getting better bioethanol yields, fermentation time is also reduced that makes it a cost effective technique.. xv.

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(16) Resumo BAGAÇO DE LARANJA COMO BIOMASSA PARA PRODUÇÃO DE ETANOL-2G Os biocombustíveis de segunda geração surgiram como fontes energéticas promissoras, podendo ser obtidos a partir de vários tipos de biomassa que não seja utilizada para alimentos. Um tipo de biomassa que apresenta baixo custo além de apresentar níveis elevados de carboidratos, é a biomassa obtida após o processamento da laranja (Citrus processing waste from oranges, CPWO). Há um grande interesse na exploração desta biomassa em termos da produção do bioetanol (etanol da 2G). Nosso trabalho visa melhorar os processos de hidrólise do CPWO comparando o rendimento do processo clássico de hidrólise ácida com aplicação de enzimas comerciais ou provenientes do microrganismo Xanthomonas axonopodis pv. citri, cepa 306 (um fitopatógeno). Os resultados obtidos com a presente investigação evidenciam que ocorreu a conversão bem-sucedida do CPWO em uma mistura de açúcares. A posteriori, os açúcares redutores que foram obtidos foram convertidos em bioetanol por meio da fermentação em mono- e co-cultura. Para tanto, foi empregada a espécie Saccharomyces cerevisiae e duas cepas de Candida parapsilosis IFM 48375 e NRRL Y-12969, sendo que as duas últimas foram isoladas a partir do bagaço da laranja. Os rendimentos em termos de bioetanol obtido nas fermentações aplicando co-culturas estavam ao redor de 50 a 62%, constituindo valores muito maiores comparados com os obtidos por cepas usadas individualmente. Além disso, os açúcares foram consumidos mais rapidamente (6 h), tornando tais processos atraentes em termos de custo e aplicações comerciais. xvii.

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(18) INDEX LIST OF ABBREVIATIONS……………………………………………………....…xxi LIST OF TABLES……………………………………………………………….…...xxiii LIST OF FIGURES………………………………………………………………...…xxv LIST OF SCHEMES………………………………………………………………..xxviii CHAPTER 1 – INTRODUCTION……………………………………………………...1 1.1. Motivation……………………………………………………………………..3 1.2. Orange Fruit…………………………………………………………………...7 1.3. CPWO Composition ………………………………………………………….8 1.3.1. Essential oil………………………………………………………………10 1.3.2. Hesperidin………………………………………………………………..11 1.3.3. Lignocellulosic fibers……………………………………………………...13 1.4. Pretreatment of Biomass……………………………………………………..17 1.5. Hydrolysis……………………………………………………………………19 1.5.1. Brief history of biomass hydrolysis……………………………………...20 1.5.2. Acid Hydrolysis………………………………………………………….21 1.5.3. Enzyme Hydrolysis………………………………………………………24 1.6. Xanthomonas axonopodis pv. citri (Xac 306)..................................................30 1.7. Factors affecting the enzymatic hydrolysis ………………………………….33 1.7.1. Substrate-related factors…………………………………………………...33 1.7.1.1. Cellulose Crystallinity………………………………………….33 1.7.1.2. Lignin Content…………………………………………………34 1.7.1.3. Specific surface Area…………………………………………..35 1.7.1.4. Degree of polymerization………………………………………36 1.7.2. Enzyme-related factors……………………………………………………37 1.7.2.1. End Product Inhibition…………………………………………37 1.7.2.2. Adsorption Reaction……………………………………………38 1.7.2.3. Enzyme inactivation …………………………………………...38 1.7.2.4. Synergism………………………………………………………39 1.8. Fermentation of Hydrolyzates………………………………………………….39 1.8.1. Submerged-Fermentation System………………………………………41 CHAPTER 2 – OBJECTIVES…………………………………………………………45 CHAPTER 3 – MATERIALS AND METHODS……………………………………..49 3.1. Citrus processing waste from Oranges (CPWO)……………………………...51 3.2. Primary Characterization ……………………………………………………..51 3.2.1. Determination of Total Solids…………………………………………..52 3.2.2. Determination of Moisture Contents……………………………………53 3.2.3. Determination of Ash Contents…………………………………………54 3.2.4. Determination of Pectin…………………………………………………55 3.2.5 Fiber Analysis……………………………………………………………56 3.2.5.1. Neutral Detergent Fiber (NDF)…………………………………56 3.2.5.2. Acid Detergent Fiber (ADF)…………………………………….58. xix.

(19) 3.2.5.3. Acid Detergent Lignin (ADL)…………………………………..60 3.2.6. Determination of Protein contents by Bradford…………………………61 3.3. Hesperidin ………………………………………………………………… 62 3.3.1. Extraction……………………………….………………………………62 3.3.2. Work up…………………………………………………………………63 3.3.3. Recrystallization………………………………………………………...63 3.4. Pretreatment for extracting essential oil……………………………………...64 3.5. Acid Hydrolyses ………………………………………………………………65 3.5.1. Determination of acid insoluble lignin………………………………….66 3.5.2. Determination of acid soluble lignin…………………………………….67 3.6. Enzymes used for enzymatic pretreatment of CPWO………………………...68 3.7. Protein lysates of “Xac 306” ………………………………………………….69 3.8. Enzyme activities……………………………………………………………...70 3.8.1. Pectinase and Polygalacturonase (PG) activities………………………..70 3.8.2. Beta-glucosidase Assay…………………………………………………71 3.8.3. Cellulase assay…………………………………………………………..72 3.9. Enzyme Hydrolysis……………………………………………………………74 3.10. Yeast strains and growth medium……………………………………………75 3.11. Mono- and co-culture fermentations…………………………………………76 3.12. HPLC Analysis ……………………………………………………………...77 3.13. Gas chromatography–mass spectrometry (GC-MS)…………………………77 3.14. Nuclear magnetic resonance (NMR)………………………………………...79 3.15. Infrared spectroscopy (IR)…………………………………………………...79 3.16. Statistical Analyses…………………………………………………………..80 CHAPTER 4 – RESULTS AND DISCUSSION………………………………………81 4.1. Compositional Analyses of CPWO…………………………………………...83 4.2. Extraction and characterization of essential oil from CPWO…………………86 4.3. Extraction and characterization of hesperidin…………………………………87 4.4. Hydrolyses of CPWO…………………………………………………………96 4.4.1. Acid Hydrolysis…………………………………………………………97 4.4.2. Enzyme hydrolysis involving commercial enzymes…………………102 4.4.3. Enzyme hydrolysis involving enzyme cocktails……………………….105 4.4.4. Enzyme hydrolysis involving Xac 306 enzymes………………………106 4.5. Submerged Fermentations of Hydrolyzates ……………………………..…..110 4.5.1. Results of Submerged fermentations of acid hydrolyzates ………..…..112 4.5.2. Results of Submerged fermentations of enzyme hydrolyzates ……..…118 4.5.2.1. Mono-culture fermentations……………………………..…....118 4.5.2.2. Co-culture fermentations………………………………..….…121 4.6. 2G-Ethanol quality………………………………………………………...…127 CHAPTER 5 – FINAL REMARKS…………………………………………….…....129 CHAPTER 6 – REFERENCES…………………………………………………...….133 Appendix……………………………………………………………………………….149. xx.

(20) LIST OF ABBREVIATIONS 5-HMF: 5-hydroxymethylfurfural AD: Acid detergent ADF: Acid detergent Fiber ADL: Acid detergent Lignin AOAC: Association of Official Analytical Chemists bp: Base pairs CBD: Cellulose-binding domain CBH: Cellobiohydrolase CD: Catalytic domain C.P IFM: Candida parapsilosis IFM48375 C.P NRRL: Candida parapsilosis NRRL-12969 CPWO: Citrus Processing Waste from Oranges CTAB: Cetyl trimethylammonium bromide CTBE: Brazilian Bioethanol Science and Technology Laboratory DM: Dry matter DNS: Dinitrosalicylic acid DP: Degree of polymerization DS: Degree of synergism EC: Enzyme commission/ classification EG: Endo glucanase FPU: Filter Paper Unit GA: Galacturonic acid GMO: Genetically modified organisms LB: Luria Bertani ND: Neutral Detergent NDF: Neutral Detergent Fiber NREL: National renewable energy laboratory PB sheet: parallel β sheet PNPG: p-nitrophenyl-β-D-glucopiranoside PNP: p-nitrophenol pv.: pathovar RT: Retention time S.C: Saccharomyces cerevisiae SmF: Submerged Fermentation SSF: Solid State Fermentation Str: Stretching TBI: Triple Resonance Broadband Inverse USDA: United States Department of Agriculture Xac 306: Xanthomonas axonopodis pv. citri (Xac 306) YPD: Yeast peptone dextrose. xxi.

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(22) LIST OF TABLES Table I-1. Chemical composition of orange peel in percentage on dry matter (DM) basis [Rivas et al., 2008] Table I-2. Chemical composition of orange peel (g/kg DM) [Bampidis and Robinson, 2006] Table I-3. Comparison between Concentrated- and Dilute-Acid Hydrolysis [adapted from Taherzadeh and Karimi, 2007; Galbe and Zacchi, 2002] Table III-1. Neutral detergent solution for determining NDF Table III-2. Acid detergent solution for determining ADL Table III-3. Reagents for preparing Bradford Table III-4. Preparation of DNS reagent Table III-5. Preparation of growth medium for yeast cells Table IV-1. Chemical composition of CPWO (local restaurant, Campinas) Table IV-2. Fiber analysis of CPWO Table IV-3. Chemical composition of industrial CPWO samples Table IV-4. Hesperidin 1H NMR chemical shifts and assignments Table IV-5. Hesperidin 13C NMR chemical shifts and assignments Table IV-6. Sugar concentrations obtained after acid hydrolysis of CPWO Table IV-7. Sugar concentrations obtained after acid hydrolysis of industrial CPWO Table IV-8. Sugar yields (% g g-1 dry CPWO) obtained in hydrolyses with pectinase, cellulase and beta-glucosidase after 48 h of enzyme hydrolysis Table IV-9. Sugar yields (% g g-1 dry CPWO) after 48 h of enzyme hydrolysis with enzyme cocktails Table IV-10. Sugar yield (% g g-1 dry CPWO) after 48 h of enzyme hydrolysis with Xac 306 enzymes. Table IV-11. Comparative analysis of mono-culture fermentation results obtained from enzymatic hydrolyzates Table IV-12. Comparative analysis of fermentation results obtained in mono and coculture treatments of enzymatically hydrolyzed CPWO. xxiii.

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(24) LIST OF FIGURES Figure I-1. Cellulosic feedstock for second generation biofuels (figure taken from Bioeconomics of Biofuels by Stephen Polasky. http://faculty.apec.umn.edu/spolasky/. Web accessed on October 23rd, 2012). Figure I-2. Top orange fruit country production in 2010 (figure taken from Food and Agriculture Organization of the United Nations, FAOSTAT, http://faostat.fao.org/site/339/default.aspx. Web page accessed on January 10th, 2013). Figure I-3. Anatomy of Orange (figure taken from sig biz website, http://www.sig.biz/site/en/medien/medien_/medienarchiv/news_archive_combibloc/news _detail_archive_combibloc_2313.jsp. Web page accessed on January 5th, 2013). Figure I-4. Structure of Hesperidin. Figure I-5. Three dimentional structure of plant cell wall with its primary and secondary layers (http://www.accessscience.com/search.aspx?rootID=791275. Web page accessed on January 14th, 2013). Figure I-6. Basic molecular structures of the major biopolymers forming the cell wall of biomass: cellulose, hemicellulose, pectin and lignin [Greil, 2001]. Figure I-7. Illustration of three cellulose strands. White balls are hydrogen atoms, black balls are carbon atoms, red balls are oxygen atoms, and turquoise lines are electrostatic hydrogen bonds (http://en.wikipedia.org/wiki/File:Cellulose_spacefilling_model.jpg. Web page accessed on January 12th, 2013). Figure I-8. Lignin (orange) and hemicellulose (blue) binding with cellulose (yellow) strands (Department of Energy Genomic, http://genomics.energy.gov. Web page accessed on January12th, 2013). Figure I-9. Scheme showing lignocellulosic biomass fibril structure. The lignin and hemicellulose surrounds cellulose. Pretreatment of biomass removes hemicellulose and lignin from the cellulose polymer chains before hydrolysis (adapted from Chandra et al., 2012). Figure I-10. Mechanism of acid hydrolysis [Verendel et al., 2011]. Figure I-11. Computer model of the structure of the endoglucanase showing catalytic domain during hydrolyses of internal bonds in the cellulose chain [Sarita, 2010]. Figure I-12. Endoglucanase enzyme from Trichoderma reesei (PDB: 1EG1). The arrows indicate beta-strands while the twisted ribbons are alpha-helices. http://www.rcsb.org/pdb/images/1eg1_asr_r_500.jpg. Web page accessed on January 12th, 2013).. xxv.

(25) Figure I-13. Computational model of the structure of the exoglucanase, CBH I; showing cellulose binding module and catalytic domain in the form of a tunel, during enzyme action [figure adapted from Carvalho, 2009]. Figure I-14. Schematic representation of the Cellobiohydrolase I from Trichoderma reesei (PDB: 7CEL) catalytic domain with a cellooligomer. Secondary-structure elements are colored as follows: β strands, blue, green and sea green arrows; α helices, orange, yellow and brown spirals; loop regions, orange, red, green, yellow and blue coils. The cellooligomer is shown in blue and red as a ball-and-stick model [Divne et al., 1998]. Figure. I-15. Crystal structure of β-glucosidase (PDB:3CMJ). (A) Overall structure of βglucosidase. The four loops where the substrate enters are colored in pink. (B) Representation of the surface structure. The large cleft is colored in yellow. This cleft had a cavity of approximately 15 × 10 Å; therefore the related disaccharides substrate could easily enter the active site pocket [Nam et al., 2010]. Figure I-16. Mode of cellulolytic enzyme action [adapted from Zhu, 2005; Jørgensen et al, 2007]. Figure I-17. The ribbon diagram of Aspergillus niger pectin lyase B (PDB: 1QCX). Helices are represented by pink coils and β structure is shown by arrows. The parallel β (PB) sheet, PB1, is yellow; PB2 is blue; and PB3 is red. The antiparallel β structure within the first T3 turn and a short β strand within the third T3 loop are indicated in orange [Vitali et al., 1998]. Figure I-18. (a) Citrus canker lesions caused by Xanthomonas axonopodis pv. citri on immature fruit with chlorotic halos. (b) SEM of stomata on grapefruit leaf with Xac bacteria entering stomatal chamber [Cubero et al., 2001]. (http://www.apsnet.org/publications/apsnetfeatures/Pages/citruscanker.aspx. Web page accessed on January 13th, 2013). Figure I-19. Time course for the enzymatic hydrolysis of lignocellulosic substrates [adapted from Mansfield et al., 1999]. Figure I-20. (a) Large SSF unit at Soufflet’s malting plant in Arcis-sur-Aube, France. http://www.allaboutfeed.net/ (Web page accessed on July13th, 2012); (b) Large SmF Wine Unit http://www.wine-searcher.com/m/2012/11/eucalyptus-in-australian-red-wines (Web page accessed on January 12th, 2013). Figure III-1. (a) CPWO; (b) Food processor; (c) Homogenized CPWO. Figure IV-1. (a) Gas Chromatogram of the extracted essential oil, (b) mass spectrum of the main peak in the oil extract (D-limonene). Figure IV-2. Extracted samples of (a) crude and (b) recrystallized hesperidin. Figure IV-3. Scanning electron micrographs of Hesperidin with (a) 10 µm (b) 5 µm and (iii) 1 µm scales.. xxvi.

(26) Figure IV-4. FTIR spectrum of hesperidin. Figure IV-5. 1H NMR spectrum of hesperidin. Figure IV-6. 13C NMR spectrum of hesperidin. Figure IV-7. COSY NMR spectrum of hesperidin. Figure IV-8. HSQC NMR spectrum of hesperidin. Figure IV-9. HPLC chromatogram showing profile of glucose (RT=15.131), fructose (RT=15.732), arabinose (RT=16.306), cellobiose (RT=13.732), galacturonic acid (RT=14.642), polygalacturonic acid (RT=8.823), 5-hmf (RT=33.336), furfural (RT=48.739) and ethanol (RT=25.316). Figure IV-10. (a) Acid treated (brown color) and untreated (pale color) CPWO hydrolyzates (b) HPLC vials with hydrolyzates for analyses. Figure IV-11. Dehydration products of hexoses and acid-catalyzed secondary reactions [Kuster, 1990]. Figure IV-12. Graphical representation of released 5-HMF concentration (g L-1) as a function of H2SO4 concentration (v v-1). Figure IV-13. Lyophilized Xac 306 protein extract. Figure IV-14. Photos of (a) unhydrolyzed and (b) hydrolyzed CPWO with Xac 306 enzymes. Figure IV-15. SEM images of: (a) unhydrolyzed CPWO and (b) hydrolyzed CPWO using Xac 306 enzymes. Figure IV-16. SEM micrographs of (a) Candida parapsilosis IFM 48375; (b) Saccharomyces cerevisiae; (c) Candida parapsilosis NRRL Y-12969. Figure IV-17. A plot of sugar and 2G-ethanol concentrations in relation to time in Saccharomyces cerevisiae fermentation of 0.5% v v-1 (15 min) acid hydrolyzed sample. Figure IV-18. A plot of sugar and 2G-ethanol concentrations in relation to time in Candida parapsilosis IFM 48375 fermentation of 0.5% v v-1 (15 min) acid hydrolyzed sample. Figure IV-19. A plot of sugar and 2G-ethanol concentration in relation to time in Candida parapsilosis NRRL Y-12969 fermentation of 0.5% v v-1 (15 min) acid hydrolyzed sample. Figure IV-20. Fermentation of CPWO enzymatically treated and fermented with Candida parapsilosis IFM 48375, theoretical ethanol yield was 52% in 12 h.. xxvii.

(27) Figure IV-21. Ethanol yield with respect to C-6 sugars (glucose and fructose) produced by co-culture fermentation of Xac 306 enzyme hydrolyzates with (a) yeast Saccharomyces cerevisiae and Candida parapsilosis IFM48375, and (b) Saccharomyces cerevisiae and Candida parapsilosis NRRL-12969. Figure IV-22. Ethanol yield with respect to C-6 sugars (glucose and fructose) produced using co- culture fermentation of Xac 306 enzyme hydrolyzates with Candida parapsilosis IFM48375 and NRRL-12969. Figure IV-23. Comparison of co-culture fermentations on Xac 306 hydrolyzates (where S.C = Saccharomyces cerevisiae; C.P IFM = Candida parapsilosis IFM48375; C.P NRRL = Candida parapsilosis NRRL-12969). Figure IV-24. (A) 1H NMR and (B) fermentations of CPWO hydrolyzates.. 13. C NMR spectra of 2G-ethanol obtained from. LIST OF SCHEMES Scheme I-1: Representation of overall process showing different steps involved in this work. Scheme I-2: Schematic representation showing cellulose and hemicelluloses conversion into 2G-ethanol.. xxviii.

(28) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. *. CHAPTER I- INTRODUCTION. * Figure taken from http://sustainablog.org/2011/12/community-trees-guerrilla-grafters/ (Web page accessed on January10th, 2013).. 1.

(29) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. 2. Chapter I.

(30) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. 1. INTRODUCTION 1.1. Motivation Biofuels generated from renewable resources are currently in high demand related to their prospect for being an alternative to petroleum and diesel.. Development of energy from renewable resources can provide. domestic energy supplies while reducing net greenhouse gas emissions and providing a more favorable energy balance than traditional petroleum fuel utilization [Farrell et al., 2006]. Since 2003, when "flex fuel" vehicles were introduced in the Brazilian market [Cordeiro de Melo, 2012], there was a rapid increase in consumption of bioethanol. Today, in addition to the 80% of Brazilian vehicles that consume bioethanol, small aircraft engines are also being developed that use this technology [Soccol, 2010]. On the other hand, exports of Brazilian bioethanol have increased significantly due to growing worldwide interest related to the safe use of biotechnology to reduce environmental risks [CTBE, 2012]. Biomass is an important renewable energy resource that can be efficiently used for biofuel production. Biomass can be defined as all organic matter of vegetal or animal origin, which is produced in natural or managed ecosystems (agriculture, aquaculture, forestry) [Vandame, 2009]. The most common fuels derived from biomass are ethanol, biodiesel and biogas. Biofuels are theoretically carbon neutral, which means that the carbon dioxide gas emissions after biofuel combustion are captured again by the plants. Biofuels can be classified as first, second and third generation, according to the origin and biomass processing. 3.

(31) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Today, most biofuels in use are first generation biofuels that are derived from food crops such as seeds or grains from cereals, sugarcane etc. This situation has triggered growing concerns over future food shortages and increasing food prices [Science business report, 2011]. To solve this problem, second generation biofuels have come forth, from non-food crops or organic waste materials that comes from food wastes, manure and agricultural residues (Figure I-1).. Figure I-1. Cellulosic feedstock for second generation biofuels (figure taken from Bioeconomics of Biofuels by Stephen Polasky. http://faculty.apec.umn.edu/spolasky/. Web accessed on October 23rd, 2012).. Conventionally, sugarcane bagasse is used as a raw material to produce second generation bioethanol [Soccol et al, 2010], but other agroindustrial wastes can also be treated, such as citrus processing waste from oranges (CPWO). Citrus fruits are among the most popular fruits grown and consumed all over the world. From the past many years, Brazil has shown significant citrus fruit production (Figure I-2) and Brazilian orange juice. 4.

(32) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. factories generated millions of tons of CPWO annually whose disposal was a big issue.. Figure I-2. Top orange fruit country production in 2010 (figure taken from Food and Agriculture Organization of the United Nations, FAOSTAT, http://faostat.fao.org/site/339/default.aspx. Web page accessed on January 10th, 2013).. Citrus fruits (oranges, tangerines, grapefruits and lemons) and their juices are the most widely consumed. According to the U.S. Department of Agriculture (United States Department of Agriculture), Brazil is the largest producer of oranges in the world, while the United States ranks second [USDA, 2012], both of them contribute to 90% of the world's orange juice production [USDA, 2012; Zvaigzne, 2009]. In 2011, about 19 million tons of oranges were produced in Brazil, of which 15 million were generated only in the State of Sao Paulo [IBGE, 2011]. As almost 99% of the fruit from this region is processed for export, it is the overwhelming giant in worldwide orange juice production. Orange juice is traded internationally in the form of frozen concentrated orange juice to reduce the volume used, so that storage. 5.

(33) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. and transportation costs are lower [Citrus production report Wikipedia, 2013]. After extraction of the juice, about 50-60% of the fruit is left as residue, which consists of: membranes, seeds and peel [Grohman and Baldwin, 1992; Wilkins et al., 2005; Wilkins et al., 2007a]. This type of lignocellulosic waste raises a number of issues for the orange juice industries in Brazil [Bansal et al., 2011]. These wastes are sometimes used in the production of cattle feed in the form of citrus pulp pellets, but this is not economically attractive in an industrial set up [Wilkins et al., 2007a]. CPWO is rich in soluble and insoluble carbohydrates, with a small proportion of lignin [Grohmann et al., 1995]. There is a great need to explore this residue in a more attractive, inexpensive process without any environmental hazards [Edwards and Doran-Peterson, 2012], such as production of second generation bioethanol. The composition of orange peel shows that it is rich in fermentable sugars, that is, glucose, fructose, and sucrose, along with insoluble polysaccharide cellulose and pectin [Grohmann et al., 1995]. The presence of low lignin levels in comparison to other lignocellulosic biomass makes such substrates ideal for fermentationbased products, such as ethanol production; however, the presence of pectin requires either harsh pretreatment or application of enzymes for the fermentable sugar release [Grohmann et al., 1995]. Acidic and enzymatic hydrolysis of CPWO which enables the breakdown of complex carbohydrate polymers is known in the literature [Wilkins et al., 2005]. But the composition of CPWO varies significantly depending on the nature of the substrate (where it is grown) and hence changes enzyme response to substrate and also the composition of hydrolyzates. In the present work, Brazilian CPWO is explored, first by. 6.

(34) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. applying classical acid hydrolysis and later comparing it with enzyme hydrolysis. Previous studies conducted by our research group focused on the microorganism Xanthomonas axonopodis pv. citri (Xac 306), a potent gramnegative bacterial pathogen, responsible for Citrus canker disease [Tasic et al., 2007; Khater et al., 2007; Fattori et al., 2011]. This bacterium is able to penetrate the stomata, and provoke visible and circular spotted wounds [Brunings and Gabriel, 2003]. Xac 306 genome was completely sequenced in 2002 by da Silva et al. and it shows presence of genes corresponding to several hydrolytic enzymes that can help in biomass degradation. Literature studies show that yeast strains, other than Saccharomyces cerevisiae, contribute to the enhanced flavor and aroma of wine because of the greater quantity of secondary metabolites produced [Garde-Cerdán and Ancín-Azpilicueta, 2006, Moreira et al., 2008]. Although Saccharomyces cerevisiae strain is more effective for the fermentation, it yields lower levels of aromatic compounds [Andorrà et al., 2012]. That is why it is important to explore the non-Saccharomyces strains not only to check their ability to ferment, but also to test the strains that might be tested further in wine production.. 1.2. Orange Fruit Orange trees are widely cultivated in tropical and subtropical climates for their delicious sweet fruit. The orange fruit is composed of the following fundamental parts (Figure. I-3): i) the exocarp called flavedo (external colored part of the peel ),. 7.

(35) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. ii) the mesocarp called albedo (white internal part of the peel), iii) the endocarp pulp, subdivided into segments containing vesicles filled with juice and seeds. Figure I-3. Anatomy of Orange (figure taken from sig biz website, http://www.sig.biz/site/en/medien/medien_/medienarchiv/news_archive_combibloc/news_detail_ archive_combibloc_2313.jsp. Web page accessed on January 5th, 2013).. 1.3. CPWO Composition In order to harness the maximum value from CPWO, it is essential to have knowledge regarding its chemical composition [Rivas et al., 2008; Ali et al., 2010; Bermejo et al., 2011; Asikin et al., 2012]. Briefly CPWO contains 75-82% moisture contents, soluble sugars, fibers (including cellulose, hemicellulose, lignin and pectin) along with ash, fats and proteins [Ali et al., 2010]. The quantitative composition determined by Rivas et al. (2008) is detailed in Table I-1:. 8.

(36) Chapter I. ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. The small quantities not identified (about 4.35% of the peel) include organic acids such as citric acid, malic acid, malonic acid, oxalic acid (which collectively represent about 1%) and vitamins such as Vitamin C (ascorbic acid). Table I-1. Chemical composition of orange peel in percentage on dry matter (DM) basis [Rivas et al., 2008] Soluble. Starch. Cellulose. sugars (%). Lignin. Pectin. Ash. Fat. Protein. Other components. (%). (%). 16.90. Hemicellulose. (%). (%). (%). (%). (%). (%). 3.75. 9.21. (%). 10.50. 0.84. 42.50. 3.50. 1.95. 6.50. 4.35. Bampidis and Robinson (2006) also investigated the composition of orange peel, and reported that the dry matter (DM) content of orange peel is mainly organic, containing proteins and other short-chain (no more than four carbons) organic acids (Table I-2). Table I-2. Chemical composition of orange peel (g/kg DM ) [Bampidis and Robinson, 2006] Organic. Crude. Neutral. Acid. Lactic. Acetic. Propionic. Isobutyric. matter. protein. detergent. detergent. acid. acid. acid. acid. fibre. fibre. (g/kg). (g/kg). (g/kg). (g/kg). (g/kg). (g/kg). 23. 20. 0.3. 0.6. 975. 58. (g/kg). (g/kg). 200. 129. Calcium. Phosphate. (g/kg). (g/kg). 7.3. 1.7. pH. 3.64. One kilogram of orange peel analyzed in these analyses had 233g DM.. It is important to note that just like all other plants products, the chemical composition of oranges varies. It is affected by various factors such as growing conditions, fruit maturity, rootstock, variety, and climate 9.

(37) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. [Kale and Adsule, 1995]. The pH of citrus peel is also variable and it can be as low as 3.64. It should be tested before any application as neutralization might be required. Bermejo et al. (2011) did analysis of bioactive agents in citrus varieties. They observed that rind contents of different varieties showed similar tendencies for the most of compounds like the flavanone glycosides hesperidin and narirutin, carotenoid and β-cryptoxanthin. Limonene was the most abundant terpene found in peel essential oil in all cultivars studied, followed by myrcene. Calcium and potassium were the dominant macronutrients. CPWO composition (as shown in Tables I-1 and I-2), show CPWO potential to be employed in different applications. Extraction of highly valuable natural products, limonene and hesperidin for example, could transform what is typically considered to be a problematic substrate to a high value commodity. Among these, pectin is also very attractive, as well as, fermentable sugars that can be used to produce bioethanol.. 1.3.1.. Essential oil. Citrus fruit contains essential oil, located in oil sacs or glands that range in diameter from 0.4 to 0.6 mm. They are present in irregular depths in the flavedo located at the outer rind of the fruit. Merle et al. (2004) studied different varieties of citrus and identified that 97% of the oil content is limonene. D-limonene (C10H16) is a hydrocarbon, classified as a cyclic. 10.

(38) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. terpene. It is colorless liquid at room temperature, with an extremely strong orange odor. The extraordinarily high amount of limonene that accumulates in orange oil glands suggests an important biological role for this terpene compound in fruit aroma and in the plant’s interactions with the environment. It has a defensive function to makes fruit resistant not only to fungal and bacterial citrus pathogens, but it is also known to function as an insect repellent [Ibrahim et al., 2001], repellent of some birds like Starlings [Clark, 1997] and mammals such as deer [Vourc'h et al., 2002]. D-limonene is employed in chemical industry, cosmetics, domestic household products and as a flavoring agent in food and medicine [Smyth and Lambert, 1998].. 1.3.2. Hesperidin The flavonoids are secondary plant metabolites that belong to the class of plant phenolics. Currently, there is a great interest in these compounds because of interesting biological activities [Mazzaferro and Breccia, 2012]. Moreover, they are abundant in the by-products, mostly in peels (albedo + flavedo), accounting for 4–12% dry weight of fruit not being used [Marín et al., 2007]. In citrus varieties, the most abundant flavonoid is hesperidin (C28H34O15). Its aglycon form is called hesperetin.. 11.

(39) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Figure I-4. Structure of hesperidin.. Hesperidin (Figure I-4) is believed to play a role in plant defense. Researchers have shown that there is a close connection between fruit growth and content of hesperidin. Also harvest time effect hesperidin and other flavonoid contents of fruit peel. Del Rio et al. (1995) showed that highest level of hesperidin accumulated in very young tissues inside the fruit, which indicates the protective function of flavonoids. Hesperidin is of great biological and medicinal interest as it is associated with lowered risk of certain cancers, reduced cholesterol and stroke. Moreover, antioxidant, anti-inflammatory, antimicrobial and antiviral properties have also been demonstrated both ‘in vitro’ and ‘in vivo’ [Cano et al., 2008; Mazzaferro and Breccia, 2012]. Hesperidin can be isolated by variety of ways using both analytical and preparative techniques. Its extraction from CPWO is very important as it can convert an agricultural waste into a valuable commodity.. 12.

(40) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. 1.3.3.. Chapter I. Lignocellulosic fibers. Cell wall is a characteristic feature of all plant cells and is rich in polysaccharides. The structural material in the cell wall is known as lignocelluloses, which provides support, strength and shape to the plant. It mainly contains: pectin, cellulose, hemicelluloses and lignin [Wyman, 1996]. Different parts of the plant have different proportion of these components. The outer wall is mainly composed of lignin that is called primary cell wall. It surrounds growing and dividing plant cells. Next is the much thicker and stronger secondary cell wall (Figure I-5), which accounts for most of the carbohydrate part in biomass [Carpita et al., 2001]. The secondary wall usually consists of three sub-layers, which are termed as S1 (outer), S2 (middle), and S3 (inner), respectively. The formation of these three distinctive layers in secondary cell walls is a result of changes in the orientation of cellulose microfibrils during their deposition. The S1 and S3 layers are typically thin and have cellulose microfibrils oriented in a flat helix relative to the elongation axis of the cell [Zhong and Ye, 2009]. The S2 layer is thick and has cellulose micro-fibrils that accounts for 75% to 85% of the total thickness of the cell wall [Plomion et al., 2001]. It is the S2 layer that largely determines the mechanical strength of fibres in wood. Hemicelluloses and lignin are also present in each of these layers [Plomion et al., 2001; Zhao et al., 2012].. 13.

(41) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Figure I-5. Three dimentional structure of plant cell wall with its primary and secondary layers (http://www.accessscience.com/search.aspx?rootID=791275. Web page accessed on January 14th, 2013).. Pectin is an important component of cell wall and is a polysaccharide rich in galacturonic acid and galacturonic acid methyl ester units (Figure I6). As a soluble fiber and combined with proteins and other polysaccharides, pectin forms skeletal tissue that makes plant chemically stable and physically strong [Willats et al. 2006]. Cellulose (Figure I-6) is a high molecular weight linear insoluble polymer of D-glucose units connected via β (1,4) glycosidic bonds. Cellulose is usually found in close association with hemicellulose and lignin. In most cases cellulose does not occur alone in a free threadlike chain, but is present in a bundle of fibrillar units with a supramolecular structure consisting of crystalline and amorphous regions [Mansfield et al., 1999]. Typically, the crystalline cellulosic region consists of several sheets of long chain fibers arranged by both intra- and intermolecular hydrogen bonds as shown in Figure I-7 [Mansfield et al., 1999].. 14.

(42) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Figure I-6. Basic molecular structures of the major biopolymers forming the cell wall of biomass: cellulose, hemicellulose, pectin and lignin [Greil, 2001].. Figure I-7. Illustration of three cellulose strands. White balls are hydrogen atoms, black balls are carbon atoms, red balls are oxygen atoms, and turquoise lines are electrostatic hydrogen bonds. (http://en.wikipedia.org/wiki/File:Cellulose_spacefilling_model.jpg. Web page accessed on January 12th, 2013).. 15.

(43) Chapter I. ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. The structure of cellulose can be cleaved by hydrolysis of β (1→4) glycosidic bond catalyzed with acids or cellulase. However, the hydrogen bonds present in long chains of cellulose in a crystalline structure (Figure I7), prevents hydrolysis and thus harsh pre-treatment conditions are sometimes required [Gardner and Blackwel, 1974]. It is widely accepted that the higher the crystalline content of cellulose, the more difficult is the enzymatic attack to hydrolyze this polymer. The amorphous regions are more accessible to enzymes and are therefore more easily hydrolyzed, while in the crystalline areas enzyme contact efficiency is decreased [Chang et al., 2000; Carvalho, 2009]. Hemicellulose (Figure I-6) is a heteropolysaccharide of arabinose, galactose, glucose, mannose and xylose, and can also contain minor amounts of other compounds, such as acetic acid. Unlike cellulose, hemicellulose polymers. are. chemically. heterogeneous,. have. lower. degrees. of. polymerization and are mostly amorphous. That is why, hemicellulose chains are more easily hydrolyzed into monomeric units as compared to cellulose chains [Wyman, 1999]. There are many suitable pretreatments for removing hemicellulose, which greatly facilitates subsequent cellulose digestion [Saha et al., 2005]. Lignin (Figure I-6) is a cross linked macromolecular material based on a phenylpropanoid monomer structure [Doherty et al., 2011]. Lignin adds strength and rigidity to cell walls and is more resistant to enzyme attack than cellulose and other structural polysaccharides [ Kirk, 1971; Baurhoo et al., 2008]. Lignin is able to form covalent bonds with hemicellulose (Figure I-8). Covalent bonds between lignin and carbohydrates mostly consist of benzyl. 16.

(44) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. esters, benzyl ethers and phenyl glycosides [Smook, 2002]. In this way, lignin provides integrity, structural rigidity and prevention of swelling of lignocelluloses. It is commonly accepted as one of the major factors responsible for biomass recalcitrance to enzymatic hydrolysis. It implies steric hindrance and prevention of fiber swelling, the later being an important factor to increase internal surface area [Mooney et al., 1998; Carvalho, 2009].. Figure I-8. Lignin (orange) and hemicellulose (blue) binding with cellulose (yellow) strands (Department of Energy Genomic, http://genomics.energy.gov. Web page accessed on January12th, 2013).. 1.4. Pretreatment of Biomass It is known that porosity (accessible surface area) of the waste materials, cellulose with high crystallinity, along with high lignin and hemicellulose content affect the hydrolysis of cellulose [McMillan, 1994]. Thus, to achieve high yields of glucose, lignocellulose must be pretreated first. The effect of pretreatment of lignocellulosic materials has been recognized for a long time [McMillan, 1994; Sun and Cheng, 2002]. A simplified representation of lignocellulose pretreatment is shown in the Figure I-9.. 17.

(45) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. The main objectives of pretreatment are: i) to reduce cellulose crystallinity, ii) to remove lignin, iii) to increase the porosity of the materials, and iv) to make substrate significantly more susceptible to enzyme action.. Figure I-9. Scheme showing lignocellulosic biomass fibril structure. The lignin and hemicellulose surrounds cellulose. Pretreatment of biomass removes hemicellulose and lignin from the cellulose polymer chains before hydrolysis [adapted from Chandra et al., 2012].. Pretreatment must meet the following requirements [Sun and Cheng, 2002]: i). improve the formation of fermentable sugars,. ii). avoid the degradation or loss of carbohydrate,. iii). retain nearly all of the cellulose present in the original material,. iv) avoid the formation of byproducts that are inhibitory to the subsequent hydrolysis and fermentation processes, and v). be cost-effective.. 18.

(46) Chapter I. ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Physical, physico-chemical, chemical, and biological processes have been used in pretreatments of lignocellulosic materials. Typical processes include hot water, dilute acids, steam explosion, ammonia fiber explosion (AFEX), strong alkali process, as well as mechanical treatment such as hammer and ball milling are conventionally used to treat lignocellulosic biomass. In this work, autohydrolysis and limonene pretreatments were executed before hydrolysis step. The main steps in the overall process are shown in scheme I-1:. CPWO. Pre-treatment. 2nd Distillation. 1st Distillation. Hydrolysis. Mono/co-culture fermentation. Fermentable hydrolysate. pH optimization. 2G-Ethanol. Scheme I-1: Representation of overall process showing different steps involved in this work.. 1.5. Hydrolysis After pretreatment, the carbohydrate polymers in the lignocellulosic materials need to be converted further into simple sugars before fermentation, through a process called hydrolysis. The hydrolysis reaction for cellulose conversion is given in Reaction I-1. (C6H10O5)n. +. nH2O. nC6H12O6 (Reaction I-1). 19.

(47) Chapter I. ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Cellulose and hemicelluloses are converted first into simple sugars and then to 2G-ethanol by fermentation, while lignin remains as a byproduct (Scheme I-2):. Cellulose. Hemicellulose. Hydrolysis. Hydrolysis. Glucose. Fermentation. Pentose and Hexose. 2G-Ethanol. Fermentation. 2G-Ethanol. Scheme I-2. Schematic representation showing cellulose and hemicelluloses conversion into 2G-ethanol.. There are several possible methods to hydrolyze lignocelluloses. The most commonly applied methods are acidic and enzymatic hydrolyses. In addition, there are some other hydrolyses methods in which no chemicals or enzymes are applied. For instance, lignocelluloses may be hydrolyzed using gamma-ray or electron-beam irradiation, or microwave irradiation. However, these processes are far from being commercially applicable [Taherzadeh and Karimi, 2007].. 1.5.1.Brief history of biomass hydrolysis Acid hydrolysis of plant lignocellulosic biomass has been known since 1819 [Galbe and Zacchi, 2002]. However, enzymes for biomass hydrolysis can barely account for 80 years of employment and research. The search for biological causes of cellulose hydrolysis did not begin until World War II. The U.S. Army started a basic research program to understand the. 20.

(48) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. causes of deterioration of military clothing and equipment in the jungles. Out of this effort to screen thousands of samples collected from the jungle came the identification of one of the most important organisms in the development of cellulase enzymes, called Trichoderma reesei. The research was started to find out the causes of deterioration of cellulosic materials but later in early 1970’s transformed to look at cellulases as means for converting solid waste into food and energy products [US department of energy website, 2013].. 1.5.2. Acid Hydrolysis Acid Hydrolysis is a type of chemical hydrolysis that involves exposure of lignocellulosic materials to an acid for a specific period of time at specific conditions such as temperatures and pressures [Deejing et al., 2009], that results in the release of monosaccharides from cellulose and hemicellulose [Panagiotopoulos et al., 2012]. Acid hydrolysis of plant lignocellulosic biomass has been known for almost 200 years [Galbe and Zacchi, 2002]. Later, commercial scale plants were also started as for example, the modified Bergius process (40% HCl for 1 hour at 35⁰C) operated during World War II in Germany, and more recently, modified Scholler processes (0.4% H2SO4) in the former Soviet Union, Japan and Brazil [Keller, 1996]. Acid hydrolysis can be performed with several types of. acids, including. sulfurous,. sulfuric, hydrochloric, hydrofluoric,. phosphoric, nitric and formic. These acids may be either concentrated or diluted [Galbe and Zacchi, 2002; Lenihan et al., 2010]. Literature shows that. 21.

(49) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. most commonly used acid is Sulfuric acid [Harris et al., 1945; Oberoi et al., 2010; Panagiotopoulos et al., 2012]. The mechanism of acid hydrolysis is shown in Figure I-10 [Verendel et al., 2011]. Acid hydrolysis of cellulose chain begins with the protonation of the glycosidic oxygen with subsequent cleavage of C1-O bond [Mendgen and Deising, 1993; Daniel, 1994]. Delocalization of existing pair of electrons on the oxygen of the glycosidic ring occurs adjacent to C1. In the next step, the nucleophilic attack of water on C1 results in acid regeneration that terminates the depolymerization step (when hydrolysis occurs within the cellulose chain to generate new terminals) or glucose production (when hydrolysis occurs directly at the terminals) [Ogeda and Petri, 2010].. Figure I-10. Mechanism of acid hydrolysis [Verendel et al., 2011].. Acid Hydrolysis can be performed using concentrated or dilute acid. A comparison between these two cases is given in Table I-3: In economical aspects, concentrated acid hydrolysis is comparatively expensive process. Due to large amount of acids used, problems are. 22.

(50) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. associated with equipment corrosion and energy-demanding acid recovery. So, there is no remarkable on-going development of concentrated acid hydrolysis of softwood.. Table I-3. Comparison between Concentrated- and Dilute-Acid Hydrolysis [adapted from Galbe and Zacchi, 2002; Taherzadeh and Karimi, 2007] Hydrolysis Method. Advantages i) Operate at low. Concentrated-acid process. temperature ii) High sugar yield. Disadvantages i) High acid consumption ii) Equipment corrosion iii) High energy consumption for acid recovery iv) Longer reaction time (26 h). Dilute-acid process. i) Low acid. i) Operated at high. consumption. temperature. ii) Short residence. ii) Low sugar yield. time. iii) Equipment corrosion iv) Formation of undesirable by-products v) Sugar degradation vi) Inhibition of fermentation because of degradation products. 23.

(51) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. 1.5.3.Enzyme Hydrolysis Over the past few years scientists from all over the world have been shown their interest and efforts in the area of biomass enzymatic hydrolysis. Enzymes application to biomass has shown distinct advantages over acid based hydrolysis methods, for example, it occurs at a very mild process conditions, give potentially higher yields and has no corrosion problems. Therefore, enzyme hydrolysis is considered most suitable one for future 2Gethanol production from biomass [Duff and Murray, 1996; Hsu, 1996]. In this process, the long chained polymers of cellulose and hemicellulose are consumed by enzymes and monomers, hexoses and pentoses are released [Stewart et al., 2006; Stewart et al., 2008; Oberoi et al., 2010]. The CPWO can be hydrolyzed to monosaccharides by applying a combination of enzymes: pectinase, cellulase and beta-glucosidase. Both bacteria and fungi can produce cellulases for the hydrolysis of lignocellulosic material. Compared with fungi, cellulolytic bacteria produce low amounts of cellulolytically active enzymes [Sternberg, 1976; Fan et al., 1987; Duff and Murray, 1996]. Of all the fungal genera, Trichoderma has been most extensively studied for cellulase production [Sternberg, 1976]. It produces a complex mixture of cellulase enzymes with high specificity towards β-1,4 -glucosidic bonds. Cellulases are usually a mixture of various cellobiohydrolases and endoglucanases supplemented with beta-glucosidase [Sun and Cheng, 2002]:. 24.

(52) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Endoglucanase (EG, endo-1,4-D-glucanohydrolase, or EC 3.2.1.4.) attacks regions of low crystallinity in the cellulose fiber, creating free chainends. Figure I-11 shows endoglucanase that is hydrolyzing internal bonds in the cellulose chain and break intermolecular bonds between adjacent cellulose chains. They act mainly on the amorphous parts and cleave glucosidic bonds randomly, generating soluble carbohydrate chains with low degrees of polymerization [Sun and Cheng, 2002; Kumar et al, 2008].. Figure I-11. Computer model of the structure of the endoglucanase showing catalytic domain during hydrolyses of internal bonds in the cellulose chain [Sarita, 2010].. Figure I-12 shows an endoglucanase enzyme from Trichoderma reesei (PDB: 1EG1). The arrows indicate beta-strands while the twisted ribbons are alpha-helices.. 25.

(53) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Figure I-12. Endoglucanase enzyme from Trichoderma reesei (PDB: 1EG1). The arrows indicate beta-strands while the twisted ribbons are alpha-helices. http://www.rcsb.org/pdb/images/1eg1_asr_r_500.jpg. Web page accessed on January 12th, 2013).. Exoglucanase. or. cellobiohydrolase. (CBH,. 1,4-β-D-glucan. cellobiohydrolase, or EC 3.2.1.91.) degrades the molecule further by removing cellobiose units from the free chain-ends [Sun and Cheng, 2002]. These enzymes release mainly cellobiose, but also glucose and small cellodextrins (cellotriose). One exoglucanase can either act on the reducing or the non-reducing ends or the chains, but microorganisms often produce more than one type of exoglucanase, degrading cellulose chains from both directions [Zhang et al., 2004]. Figure I-13 shows the computational model of the structure of the exoglucanase enzyme and its action at the free ends of cellulose polymer chain. In exoglucanase enzyme, the catalytic domain is represented by a tunel of approximately 50 Å in length [Sarita, 2010] The Xray studies by Divne et al. (1998) revealed some details about three dimentional enzyme structure of exoglucanases (Cellobiohydrolase I) (Figure I-14).. 26.

(54) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Figure I-13. Computational model of the structure of the exoglucanase, CBH I; showing cellulose binding module and catalytic domain in the form of a tunel, during enzyme action [figure adapted from Carvalho, 2009].. Figure I-14. Schematic representation of the Cellobiohydrolase I from Trichoderma reesei (PDB: 7CEL) catalytic domain with a cellooligomer. Secondary-structure elements are colored as follows: β strands, blue, green and sea green arrows; α helices, orange, yellow and brown spirals; loop regions, orange, red, green, yellow and blue coils. The cellooligomer is shown in blue and red as a ball-and-stick model [Divne et al., 1998].. 27.

(55) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Beta-glucosidase (EC 3.2.1.21) hydrolyzes cellobiose that is an endproduct inhibitor of many cellulases and produces glucose monomers [Sun and Cheng, 2002]. Endo- and exoglucanases beak down larger oligosaccharides into cellobiose by primary hydrolysis, but the most important step is the action of β-glucosidases, that completes hydrolysis in a form that resulting sugar monomer (glucose) can be used further in the process of fermentation. Nam et al. (2010) explained crystal structure of beta-glucosidase (PDB:3CMJ), and proposed four loops where substrate (cellobiose) molecule is entered/captured for hydrolysis (Figure I-15).. Figure. I-15. Crystal structure of β-glucosidase (PDB:3CMJ). (A) Overall structure of βglucosidase. The four loops where the substrate enters are colored in pink. (B) Representation of the surface structure. The large cleft is colored in yellow. This cleft had a cavity of approximately 15 × 10 Å; therefore the related disaccharides substrate could easily enter the active site pocket [Nam et al., 2010].. Overall, the mode of cellulolytic enzyme action for cellulose hydrolysis can be summarized as demonstrated in Figure I-16:. 28.

(56) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Figure I-16. Mode of cellulolytic enzyme action [adapted from Zhu, 2005; Jørgensen et al, 2007].. In addition to the three major groups of cellulase enzymes, there are also a number of enzymes that attack hemicelluloses, such as glucuronidase, acetylesterase,. xylanase,. β-xylosidase,. galactomannanase. and. glucomannanase [Duff and Murray, 1996]. Hemicellulases are produced by many species of bacteria and fungi, as well as by several plants. Today, most commercial hemicellulase preparations are produced by genetically modified Trichoderma or Aspergillus strains [Mussatto and Teixeira, 2010]. Apart from cellulose and hemicelluloses, pectins are abundant in the soft tissues of citrus fruits, sugar beet pulp and apple [Numan and Bhosle, 2006]. Pectinases are one of the most widely distributed enzymes in bacteria, fungi and plants. Protopectinases, polygalacturonases, lyases and pectin esterases are among the extensively studied pectinolytic enzymes [Jayani et al., 2005].. 29.

(57) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Figure I-17 shows the three-dimensional structure of Aspergillus niger pectin lyase B (PDB: 1QCX). The ribbon diagram is illustrating the secondary structure.. Figure I-17. The ribbon diagram of Aspergillus niger pectin lyase B (PDB: 1QCX). Helices are represented by pink coils and β structure is shown by arrows. The parallel β (PB) sheet, PB1, is yellow; PB2 is blue; and PB3 is red. The antiparallel β structure within the first T3 turn and a short β strand within the third T3 loop are indicated in orange [Vitali et al., 1998].. 1.6. Xanthomonas axonopodis pv. citri (Xac 306) Xanthomonas is an important genus of plant pathogenic Gramnegative bacteria [Khater et al., 2007; Tasic et al., 2007; Jalan et al., 2011; Fattori et al., 2011; Liu et al., 2012]. A great number of citrus diseases are caused by distinct pathovars (pv.) of Xanthomonas species [Vauterin et al., 1995; Schaad et al., 2006]. Citrus canker is caused by several pathogenic variants of Xanthomonas axonopodis pv. citri (Xac). Xac strain 306 with a suspected origin in southeastern Asia causes Asiatic type canker and is the most widespread and virulent bacteria [Jalan et al., 2011].. 30.

(58) ORANGE BAGASSE AS BIOMASS FOR 2G-ETHANOL PRODUCTION. Chapter I. Like many other bacterial diseases, the pathogen enters host plant tissues through stomatas [Brunings and Gabriel, 2003; Cubero et al., 2001] and wounds [Koizumi and Kuhara, 1982]. The optimum temperature for infection falls between 20 and 30°C [Koizumi, 1977]. Bacteria multiply 3-4 log units per lesion under optimum conditions and cells may emerge from stomatal openings in as little as 5 days to provide inoculums for further disease development [Jalan et al., 2011]. The earliest symptoms on leaves appear as tiny, slightly raised blister-like lesions beginning around 9 days post-infection (Figure I-18). It produces hyperplasic and hypertrophic (corky) lesions surrounded by oily or water-soaked margins and a yellow halo on leaves, stems, and fruits [Jalan et al., 2011]. These bacterial attacks on the host tissue involves impressive arsenal of proteins, including pectinases and celullases for their hydrolytic activity. Xanthomonas axonopodis pv. citri (strain 306) genome was completely sequenced in 2002 by da Silva et al., and showed that it has one circular chromosome comprising 5,175,554 base pairs (bp), and two plasmids: pXAC33 (33,699 bp) and pXAC64 (64,920 bp). The transfer of macromolecules in to the host body by Xac is the main cause of infection. Two completely distinct and highly complex multiprotein systems are considered to mediate this transfer: the type III and the type IV secretion system [Alegria et al., 2005]. Degradative enzymes and toxins are secreted by two clusters of Type II secretion system (T2SS) that cause degradation of biomass [da Silva et al., 2002; Moreira et al., 2004].. 31.